PROJECT SUMMARY/ABSTRACT Iron is an essential nutrient, but it can be limiting in aerobic environments. At the other extreme, iron catalyzes the formation of reactive oxygen species that damages cellular components, and can contribute to the mode of killing by antibiotics. The ability of bacteria to adapt to the iron status and maintain homeostasis contribute to their success as pathogens, symbionts, and in complex ecosystems generally. This proposal addresses two related hypotheses: First, iron acquisition systems can evolve rapidly to adapt to new iron chelates present in microbial environments. Second, iron export is an essential mechanism in managing iron-dependent oxidative stress and maintaining homeostasis. Iron acquisition by siderophore-mediated systems is a well-described bacterial iron scavenging strategy. However, Bradyrhizobium japonicum and many bacterial species of biomedical relevance do not synthesize siderophores. These bacteria are wholly dependent on iron chelates from the environment, including siderophores made by other organisms (termed xenosiderophores in that context). Most bacteria cannot be cultured in the lab, and work by others identify xenosiderophores from co-habiting microbial neighbors as a missing nutrient. We show here that B. japonicum is an excellent bacterial model for studying xenosiderophore utilization. These multi-component uptake systems are regarded as highly specific, yet we demonstrate rapid evolution to adapt to a new iron chelate by single nucleotide mutation. Although novel in discovery, it is likely that facile adaptation is common in nature. Human patients receiving prolonged administration of siderophores or synthetic iron chelators to treat patients with iron overload often develop bacterial infections, suggesting adaptation within the human host under that selection pressure. Understanding bacterial iron homeostasis has focused almost exclusively on iron uptake because of its low bioavailability in aerobic environments, and thus very little known about iron export. We identified the iron exporter MbfA, and show that it is essential for managing iron-related stresses. Moreover, it is implicated in iron sensing and trafficking, which is conferred by an unusual N-terminal cytoplasmic domain. Finally, MbfA is functionally linked with iron storage, and we want to understand the basis of this. Three specific aims are proposed. Specific Aim 1: Characterize the plasticity of outer membrane receptors to acquire gain-of-function mutations that allow rapid adaptation to available iron. Specific Aim 2: Identify and characterize the periplasmic components of ferric siderophore uptake that allow rapid adaptation to available iron. Specific Aim 3: Elucidate the mechanism of the iron exporter MbfA and characterize its functional relationship with the iron storage protein bacterioferritin.